Project Summary The threat of multidrug resistant (MDR) bacterial infections has in recent years led to increased efforts in identifying novel antibacterials. These efforts have resulted in the approval of several new drug classes for Gram-positive infections; however due to the additional challenges imposed by the outer membrane found in Gram-negative bacteria, a novel broad-spectrum agent has not been introduced since 1968. Furthermore, the majority of compounds in the Gram-negative drug development pipeline are derivatives of existing antibiotics, which increases the likelihood of cross-resistance with approved treatments. Thus, there exists a critical need to identify novel classes of broad-spectrum antibiotics, particularly those with activity against MDR pathogens. One class of antibacterials is the nybomycins, which are particularly promising due to their impressive activity against wild-type and MDR S. aureus and Enterococci species in both cell culture and animal models of infection. However their use as broad-spectrum agents is limited by their inability to penetrate into Gram- negative bacteria. It is, therefore, the objective of this application to expand the nybomycin spectrum of activity by designing derivatives that possess suitable activity against both Gram-negative and Gram-positive pathogens. This work will build upon preliminary studies that recently identified the first nybomycin analogue with activity against Gram-negative bacteria. In Specific Aim 1, a collection of nybomycin derivatives will be prepared and evaluated for the ability to inhibit their biological target and accumulate in Gram-negative bacteria. The results from these studies will be used to determine how each of these factors contribute to antibacterial activity and guide the synthesis of more active derivatives. The therapeutic potential of these compounds will be determined in Specific Aim 2 by evaluating their tolerability, pharmacokinetic parameters, and in vivo efficacy in mice. The completion of these studies will have a significant impact on human health by providing a series of nybomycin analogues with significant activity against serious MDR Gram-positive and Gram-negative pathogens as well as the preliminary in vivo data to support their development as a novel drug class.